Approximately 170 million people worldwide are persistently infected with hepatitis C virus (HCV) and these individuals account for a majority of all cases of chronic liver disease (1). The public health impact of HCV is compounded by the overall low response rate to current interferon (IFN)-based therapies for treating HCV infection, underscoring the need for new therapeutic strategies to combat the HCV pandemic. HCV is a single-stranded positive sense RNA virus and member of the Flaviviridae (2). The 9.6-kilobase HCV genome encodes a single polyprotein that is post-translationally processed into at least 10 individual structural and nonstructural (NS) viral proteins, the latter of which are sufficient to support HCV RNA replication (3). Current studies support a model in which HCV infection results in assembly of the viral RNA and NS proteins into a replication complex that associates with the host cell endoplasmic reticulum (ER). Viral-directed processes convert the ER into a membranous web conducive to virus replication (4-6). The cellular co-factors and membrane constituents that contribute to assembly and maintenance of the HCV replication complex are not known.
West Nile (WN) virus is a member of the Flavivirus genus, which encompasses small spherical enveloped viruses harboring a single (+) RNA genome. Flavivirus genomic RNA is the only virus-specific mRNA in infected cells, encoding a single polyprotein, which is processed into structural and nonstructural viral proteins. Human infections with WN virus generally result in mild undifferentiated fever; however recent outbreaks of WN infection in North America, Europe and Israel have been characterized by relatively high rates of potentially fatal neurological disorders. See, Shi et al., 2002, J Virol 76, 5847-56; Yamshchikov et al., 2001, Virol 281, 294-304.
Cell membrane composition is subject to modification through the mevalonate pathway, which produces cholesterol and non-sterol isoprenoid products (7). Two of the mevalonate-derived isoprenoids, farnesyl (15 carbons) and geranylgeranyl (20 carbons), are attached to membrane proteins via formation of a cysteine thioether (7, 8). This process, called protein prenylation, targets certain proteins to cell membranes where they regulate many cellular functions, ranging from vesicle budding and fusion to growth regulation. Therapeutic control of the mevalonate pathway has proven effective for the clinical treatment of hypercholesterolemia and is achieved in part through the use of statin compounds (7, 9). Statins block mevalonate production by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase), resulting in a block in the subsequent steps of cholesterol synthesis (7, 9). At the high concentrations that are attainable in tissue culture cells, statins deplete mevalonate sufficiently to lower the cellular pools of farnesyl and geranylgeranyl pyrophosphates, which are the donors in the protein prenylation reactions (7, 10).
Here we disclose that HCV replication requires host protein prenylation. We further disclose that WN virus replication also requires host protein prenylation. We further disclose that prenylation inhibitors can be used to reduce host cell permissiveness to these and other viruses, particularly Flaviviridae, that do not have any prenylated viral proteins. The strict dependence of viral replication upon host protein prenylation provides a therapeutic approach for treating infection by these viruses.
Relevant Literature
Bordier et al. (2003, J. Clin. Invest. 112:407-414, 2003) report in vivo antiviral efficacy of prenylation inhibitors against hepatitis delta virus (HDV), building on prior work suggesting that HDV encodes farnesylated viral proteins, and that replication of this virus may be inhibited by an HMG CoA reductase inhibitor and a farnesyl transferase inhibitor. Bordier et al. suggests that targeting viral prenylation may provide a strategy for other medically important viruses, citing Glenn, J. S. (1995, Prenylation and virion morphogenesis. In The unique hepatitis delta virus. G. Dinter-Gottlieb, editor. R.C. Landes Publishing Co. Austin, Tex., USA. 83-93). This chapter suggests that CXXX prenylation boxes are found in hepatitis A virus, foot and mouth disease virus, and the white clover mosaic virus.
Glenn has issued several U.S. Patents describing HDV inhibition and further suggesting extrapolating their HDV treatment to other viruses that have prenylated proteins. For example, U.S. Pat. No. 6,159,939 suggests screening sequence banks for viral proteins containing a C-terminal CXXX box, and suggests inhibiting the prenylation of prenylated viral proteins of such viruses (col.7, lines 23-26). However, screening the recited viruses for CXXX boxes reveals that none of them contains a C-terminal CXXX box, nor do any of them encode a prenylated viral protein: reports of prenylated viral protein are limited to HDV and ECV (below).
Thome et al. (2001, JCB 152, 1115-22) report that equine herpes virus (ECV) encodes a protein (v-E10) that contains a C-terminal geranylgeranyltransferase II consensus site and that lovastatin can reduce membrane localization of v-E10. ECV is a DNA virus with distinct structure and replication mechanisms from RNA viruses such as Flaviviridae.
Gower and Graham (2001, Antimicrobial Agents and Chemotherapy 45, 1231-37) report antiviral activity of lovastatin against respiratory syncytial virus (RSV), and suggest that RSV replication is dependent on RhoA geranylgeranylation. RhoA geranylgeranylation is mediated by GGTase II, and RSV structure and replication are distinct from Flaviviridae and Picornaviridae.